New Directions for NanoLC

Although less than 5% of the total HPLC market, nanoLC is a fundamental part of proteomics research and has advanced the use of LC/MS. It is also one of the fastest growing segments of the HPLC market. NanoLC is defined by flow rates of 10–1000 nL/min. and columns with inner dimensions (ID) of 10-100 µm. Most every major HPLC manufacturer offers a nanoLC system and many of the top MS companies offer nanoLC/MS configured systems. The advantages of nanoLC include the conservation of sample, the ability to provide a flow rate compatible with ESI-MS and increased sensitivity.

The nanoLC market can be divided between dedicated vendors and general HPLC vendors that offer nanoLC systems. Companies specializing in nanoLC include Eksigent, Proxeon and Micro-Tech Scientific. Eksigent supplies its system for pairing with Applied Biosystems’ MS systems (see IBO 11/30/05), while Proxeon supplies its EASYnLC to Bruker and Varian for LC/MS systems (see IBO 11/30/07, 4/30/08). Agilent, Dionex, Hitachi High-Technologies, Shimadzu and Waters are among the HPLC vendors that offer nanoLC versions of their higher-end HPLC platforms.

In the crowded nanoLC market, each company must differentiate its system. Describing the advantages of Proxeon’s system, CEO Mårtin Winge described its ease of use. “Other systems on the market are designed for multiple applications based on modules that need to be assembled and connected with multiple tubes and lines,” he told IBO. “EASY-nLC is preconfigured to be connected directly with an MS. This has resulted in a product that is small and compact and typically only takes a couple of hours to unpack, install, and get up and running.” Introduced in 2005, the EASY-nLC features a splitless flow configuration, syringe pumps and a recommended flow range of 100–1000 nL/minute.

Also launched in 2005 was Dionex’s UltiMate 3000 nanoLC system. Based on Dionex’s UltiMate HPLC, the UltiMate 3000 nanoLC is designed for flexibility. “[T]he UltiMate 3000 nanoLC system features true nano-scale UV detection, a sample fractionation option as well as the ability to run larger ID columns up to analytical scale useful for sample prefractionation,” said Dietrich Hauffe, PhD, vice president for Corporate Marketing and Business Development at Dionex. The split-flow UltiMate 3000 nanoLC features a flow rate of 50–1,000 nL/min.

As with Waters’ ACQUITY UPLC system, the company’s splitless nanoACQUITY UPLC, introduced in 2004, is designed for separations at 10,000 psi using 1.7 µm particles, with a flow rate down to 200 nL/min. and column IDs of 75 µm–1 mm. “The ability to operate at higher pressures has allowed researchers to use longer columns, up to 25 cm, to achieve more resolution,” noted Patricia Young, PhD, senior Product Marketing manager for Waters.

Earlier this year, Eksigent introduced its own high-pressure nanoLC system, the NanoLC Ultra, for pressures of up to 10,000 psi. It offers flow rates of 50–500 nL/min. at high pressure. As with the company’s Express system, first introduced in 2002, a splitless flow control system is operated by pneumatic pumps. Feedback control is a distinguishing feature. “The way we are able to generate these nanoLC flow rate gradients directly is by means of measurement and really fast feedback to the pump that delivers the solvent. That guarantees a very accurate flow rate at any given time,” said Remco van Soest, product manager of its NanoLC HPLC systems at Eksigent.

Two of the prominent bottlenecks in proteomics research are the complexity of the sample matrix and the dynamic range of the sample’s proteins. Two strategies for purifying samples are fractionation prior to nanoLC/MS and 2D nanoLC/MS. Although 2D nanoLC is not a new approach, companies continue to refine methods and products for it. Dr. Hauffe described a new application for off-line 2D nanoLC for the UltiMate 3000. “In this application, a first-dimension separation of peptides or proteins is fractionated in a well-plate. Fractions are reinjected onto a second-dimension column with different selectivity,” he explained. “This application combines high peak capacity separations, flexibility and automation. Proteins can actually be digested in the autosampler for further downstream peptide analysis.”

However, according to Mr. Winge, the success of two-dimensional (2D) nanoLC workflows is varied. “Many users request the 2D capability because of its power to resolve more proteins in complex mixtures. The number of successful users is, however, a lot lower than the demand,” he said. According to him, the company’s standard EASY-nLC platform offers an simple method for 2D LC that requires no instrument adjustments. “The EASY-nLC offers a novel 2D-LC method that gives the same resolving power as ordinary 2D nanoLC, but it works on the standard 1D instrument.”

This year, Waters introduced another approach for 2D nanoLC, which is typically performed using an ion exchange column, followed by separation on a reverse phase column. At this year’s ASMS, Waters is introducing a new dual-gradient, 2D-LC technique. As Dr. Young explained, “[it is] based on packed columns that use reversed phase columns operated at high pH, followed by low pH to fully exploit the ionic and hydrophobic structure of biomolecules.”

The most popular application for nanoLC/MS is protein identification using either ESI-ion trap or MALDI-TOF MS systems. However, Mr. van Soest told IBO that he is now seeing more nanoLC systems paired with triple quadrupole MS for biomarker validation. “NanoLC has been used for the last 10 to 15 years for the discovery of biomarkers. . . . Now, they need to validate larger amounts of samples. People have started doing that with triple quads rather than the [ion] trap or the hybrid instruments that are used for discovery.”

Another trend, according to Mr. Winge, is the greater use of nanoLC by nonexperts. “A growing category of users is the proteomics nonexperts. We see a trend towards an increasing portion of users being cell biologists and clinical researchers.” He added, “on the other side, the proteomics centers of excellence groups are probably larger today, and better funded compared to a few years ago. Core labs is another category of users that are less dominating in the market today.”

These trends highlight the need for easy to use nanoLC systems, including turnkey nanoLC/MS systems, according to Mr. Winge. “As performance becomes a less dominating differentiator between MS suppliers, again complete solution offerings become a more important competitive factor,” he stated.

Mr. van Soest told IBO that nanoLC also needs to become more reliable. About higher pressure nanoLC, he noted: “So far, it’s a technology that’s been used by researchers who don’t mind packing their own columns and have been pretty patient in making it all work. But that’s slowly changing.”

One solution for improving nanoLC’s ease of use is microfluidic LC systems, which eliminate tubing, connections and fittings. At ASMS, Waters is previewing nanoTILE technology as part of its new TRIZAIC UPLC system. Thus, Waters joining Agilent, which introduced its nanoLC chip in 2005. “TRIZAIC UPLC technology delivers highly usable UPLC performance that will make nanoLC a readily approachable method for analyzing small sample amounts with high sensitivity,” said Dr. Young.

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